Insulin is the major hormone involved in glucose homeostasis and abnormalities in insulin action are central to the pathogenesis of diabetes mellitus. Consequently, detailed knowledge of the molecular mechanisms by which insulin interacts with target cells will lead to a better understanding of disease states where insulin action is abnormal. The objectives of this proposal are to define events in the cellular processing of receptors subsequent to insulin binding and determine how these processes may be related to insulin action in target cells. We propose to study cultured muscle cells since muscle is an important "in vivo" target tissue and since cell culture will allow us to study receptor regulation during chronic exposure to insulin. The first specific aim is to delineate the different steps in the endocytotic-intracellular pathway of insulin receptors. By covalently labeling insulin receptors with a radioactive analog of insulin and by using solube receptor binding assays, we will examine the kinetics of insulin receptor internatization, segregation from insulin, degradation, and recycling back to the membrane, and the cellular location of these proceses. Secondly, we will study the relationship between the processing of insulin receptors and the activation or deactivation of cellular responses to insulin, particularly insulin- stimulated glucose transport. More specifically, we will examine whether internalization of insulin-receptor complexes provides a signal for termination and transport deactivation. Furthermore, we will treat cells under conditions known to impair either internalization or deactivation and assess the effects on both processes. We will also examine whether insulin receptors that remain occupied by hormone and undergo recycling are capable of stimulating glucose transport. These experiments will determine whether occupied receptors are capable of generating a signal leading to stimulation of glucose transport as long as they are occupied or whether signal transmission is a discrete step related to the initial binding of insulin. The final objective is to elucidate mechanisms by which cells regulate the number of insulin receptors on the plasma membrane. Since exposure to insulin is known to influence receptor number, we will examine the effects of acute and chronic exposure to insulin on the flux of receptors through the internalization, recycling, and degradative pathways. Information gained from these studies should increase our knowledge of how cells respond to insulin and how the response may be altered in hyperinsulinemic states.